DESCRIPTION(Adapted from applicant's abstract): Stroke and brain injury are major causes of morbidity and mortality and significant economic loss. This competitive renewal application will explore novel hypotheses relating to regulation of death receptors as mediators of cell death in vivo and in vitro. Preliminary evidence suggests that a Death Inducing Signaling Complex (DISC) assembles after acute brain injury and initiates caspase activation leading to cell death. Five aims are proposed to contrast mechanisms relating to the death receptor Fas, death effector proteins, and initiator caspase-8 and -10 in vivo after acute brain injury (cerebral ischemia and brain trauma). In experimental models, caspase-8 cleavage and DISC assembly was significantly greater in trauma than ischemia, suggesting differences in cell death mechanisms between acute brain injuries. To explain these differences, we propose a novel hypothesis that TYPE I (mitochondria-independent) may be more typical of head trauma and TYPE II (mitochondria-dependent) cell death may predominate in ischemia, and this hypothesis will be tested in Bid-/- null mice. To dissect mechanisms regulating DISC assembly, we propose in vitro studies using enriched cortical neurons and oxygen-glucose deprivation (OGD). Our preliminary data indicate that adding FasL kills neurons during OGD, and cell death after OGD is reduced by caspase inhibitors. We propose to determine whether both OGD and FasL cell death can be regulated by c-FLIP, an inhibitor of caspase-8 activation, using viral vectors to overexpress the c-FLIP gene. In vitro studies will explore regulation of FasL expression by determining whether inhibition of Forkhead transcription factor (FKHRLI), which promotes FasL synthesis, reduces OGD-mediated neuronal cell death. We also propose to establish the functional relevance of Fas/FasL signaling in vivo using Fas-/- mice, antisense treatment, gld mice and overexpression of c-FLIP using HSV-I amplicon viral transfer. Finally, we will expand upon preliminary data detecting DISC assembly in human brain following acute injury and thereby validate the importance of cell surface death receptors in acute injury. Together these experiments will explore death receptor-mediated acute cell killing within brain in order to identify mechanisms and potential targets of ischemic and traumatic brain injury.